Hepatitis C virus (HCV) is a major cause of chronic hepatitis, with the complications of liver failure and cancer, throughout the world. Developing an effective vaccine has been difficult. Moreover, the identification of new antivirals has been limited by the lack of model HCV systems. Both the development of model systems to study HCV replication and identification of molecular based therapeutics represent major unsolved problems. The HCV RNA-dependent RNA polymerase (RDRP) is a likely target for HCV antivirals. We have expressed recombinant HCV RDRP and isolated it as an active enzyme. The hypothesis to be tested is that a model in vitro system can be developed to study key aspects of HCV replication and to identify molecular based therapeutics that target the HCV RNA-dependent RNA polymerase. The over-all objective of this work is to further purify and characterize the HCV RNA-dependent RNA polymerase (RDRP) and initiate studies to identify RNA binding proteins that are candidate regulatory subunits of HCV RDRP. The further purification, characterization of HCV RDRP and the identification of regulatory subunits should eventually permit more specific molecular based therapeutics for chronic hepatitis C to be developed. Specific Objectives: * To further purify and characterize the HCV RDRP. * To perform site directed mutagenesis of HCV RDRP to determine the importance of specific amino residues and to engineer an active enzyme that can be more rapidly purified. * To identify cellular or viral proteins bind the 3' genomic RNA structural motif and determine their effect on HCV RDRP activity (long- term).